Project Summary RIPK1 and RIPK2 are homologous protein kinases that play distinct roles in the activation of inflammatory signaling and, in case of RIPK1, regulated necroptotic and apoptotic cell death in response to ligands of different (TLR and NOD) families of innate immune Pattern Recognition Receptors. These kinases attracted significant interest due to their involvement in a broad range of inflammatory and necrotizing pathologies. In particular, compelling evidence supports the roles for RIPK1 and RIPK2 in the etiology of Multiple Sclerosis (MS). Analysis of human samples revealed that both RIPK1 and RIPK2-associated pathways are activated in cortical white matter lesions of MS patients. Furthermore, blocking the catalytic activities of these kinases by small molecule inhibitors produced a significant alleviation of disease in the mouse experimental autoimmune encephalomyelitis (EAE) model for MS. Additionally, inhibition of RIPK1 showed a significant benefit in the cuprizone-induced demyelination model for MS. Importance of RIPK2 was affirmed in Ripk2-/- mice, which were also resistant to the development of EAE. Further analyses revealed that RIPK1 is upregulated in activated resident microglia and may provide a causative factor in the early stages of disease development following mouse immunization with MOG peptide. Conversely, RIPK2 was found to be an important signaling molecule in antigen-presenting myeloid-derived dendritic cells accumulating in the CNS. Thus, RIPK2 kinase activity may control a critical link between EAE-associated innate and adaptive immune responses. These findings led to our central hypothesis that kinase activities of RIPK1 and RIPK2 act cooperatively through activation of distinct neuroinflammatory mechanisms in different innate immune cell populations to drive the development of the demyelinating pathology. In the current proposal, we seek to analyze the time course of protection in EAE model upon co-administration of RIPK1 and RIPK2 inhibitors. In particularly, in Aim 1, we will evaluate mono vs. co-administration of RIPK1 and RIPK2 inhibitors to elucidate to what time point this approach remains efficacious. Another objective that we seek to address is initial evaluation of RIPK1 and RIPK2 pathway activation in different cell lineages in the spinal cord to begin understanding the functional roles and co-roles of these pathways in disease progression. We have recently developed new classes of highly potent RIPK2 inhibitors. In Aim 2, we plan to evaluate and optimize pharmacologic properties of these molecules for in vivo CNS exposure to select optimal leads for advancement into EAE studies. Overall, our project will provide critical data revealing the feasibility of targeting RIPK1 and RIPK2 kinases as a new disease-modifying strategy for the treatment of MS.